Journal: Anatolian Journal of Cardiology

Article Title: Hypoxia-Induced Sarcoplasmic Reticulum Ca 2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes

doi: 10.5152/AnatolJCardiol.2022.1223

Figure Lengend Snippet: The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a gene expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a gene expression. n = 6, * P < .050 for comparison with 1% O 2 , # P < .050 for comparison with Vehicle, $ P < .050 for comparison with 0.3 mM (all 1-way ANOVA with Tukey post hoc test).

Article Snippet: After blocking, the membranes were washed using Tris-buffered saline with Tween (TBS-T) and then incubated with primary antibody SERCA2a (A010-23S, Badrilla, Leeds, UK, dilution 1:1000), RyR2 (ARR-002, Alomone Labs, Jerusalem, Israel, dilution 1:1000) and β-tubulin (ab6046, Abcam, Cambridge, UK, dilution 1:1000) overnight.

Techniques: Expressing

Journal: Anatolian Journal of Cardiology

Article Title: Hypoxia-Induced Sarcoplasmic Reticulum Ca 2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes

doi: 10.5152/AnatolJCardiol.2022.1223

Figure Lengend Snippet: The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a protein expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a protein expression. n = 6. No statistically significant comparison.

Article Snippet: After blocking, the membranes were washed using Tris-buffered saline with Tween (TBS-T) and then incubated with primary antibody SERCA2a (A010-23S, Badrilla, Leeds, UK, dilution 1:1000), RyR2 (ARR-002, Alomone Labs, Jerusalem, Israel, dilution 1:1000) and β-tubulin (ab6046, Abcam, Cambridge, UK, dilution 1:1000) overnight.

Techniques: Expressing

Journal: Frontiers in Cardiovascular Medicine

Article Title: Matricellular Protein CCN5 Gene Transfer Ameliorates Cardiac and Skeletal Dysfunction in mdx/utrn (±) Haploinsufficient Mice by Reducing Fibrosis and Upregulating Utrophin Expression

doi: 10.3389/fcvm.2022.763544

Figure Lengend Snippet: CCN5 prevents cardiac fibrosis in mdx/utrn (±) mice. (A) Experimental scheme for panels (B–E) . mdx/utrn (±) mice were injected with AAV.9-Con or CCN5 into the tail vein, and hearts were harvested for experiments 8 weeks later. Age-matched WT mice are shown in comparison. (B) Hearts were sectioned and stained with trichrome. Blue areas indicate fibrotic tissue and red areas indicate normal tissue. (C) The ratio of fibrotic area over total tissue of the stained hearts was plotted. (D) Proteins obtained from cardiac tissue were immunoblotted with antibodies against CCN5, α-SMA, collagen I, SERCA2a, RyR2, NCX1, phosphorylated phospholamban (p-PLN), t-PLN and α-tubulin. (E) Protein bands on western blots were scanned and plotted. n = 6. * p < 0.05 and ** p < 0.01.

Article Snippet: Transferred blots were blocked with 5% non-fat skim milk and incubated with antibodies against mouse monoclonal CCN5 (1:1,000, Sigma, #WH0008839M9), mouse monoclonal α-SMA (1:1,000, Sigma-Aldrich, #A5228), goat polyclonal collagen I (1:1,000, Abcam, ab34710), rabbit polyclonal SERCA2a (1:1,000, a custom antibody from 21st Century Biochemicals), rabbit polyclonal RyR2 (1:1,000, Alomone Lab, ARR-002), rabbit monoclonal NCX1 (1:1,000, Abcam, EPR12739), rabbit polyclonal p-PLN (1:1,000, Badrilla, A010-12AP), mouse monoclonal t-PLN (1:1,000, Badrilla, A010-14), and mouse monoclonal α-tubulin (1:3,000, Santa Cruz, #sc-8035) for 12–16 h at 4°C.

Techniques: Injection, Staining, Western Blot

Journal: eLife

Article Title: STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells

doi: 10.7554/eLife.70278

Figure Lengend Snippet:

Article Snippet: Cells were then blocked with 50% SEABLOCK blocking buffer (Thermo Fisher Scientific) for 2 hr and incubated overnight at 4°C with primary antibody (anti-STIM1- [4916], Cell Signaling Technologies, Danvers, MA; anti-STIM1 [610954], BD Biosciences, Franklin Lakes, NJ; anti-BKα1- [APC-021], Alomone Labs, Jerusalem, Israel; anti-RyR2- [MA3-916], Thermo Fisher Scientific; anti-TRPM4- (ABIN572220); https://antibodies-online.com , Limerick, PA; anti-IP 3 R- (ab5804), Abcam, Cambridge, UK) diluted in PBS containing 20% SEABLOCK, 1% BSA, and 0.05% Triton X-100.

Techniques: Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Software

Journal: iScience

Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

doi: 10.1016/j.isci.2021.103693

Figure Lengend Snippet: Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 [RyR2]), MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.

Article Snippet: For experiments in , cells infected with the different ICU3 biosensors were incubated with an anti-caveolin 3 antibody (1:200, Thermo-Fisher Scientific PA1-066) to label the PM, an anti-ryanodine receptor 2 (RyR2) antibody (1:200, Alomone Labs ARR-002) to label the SR, an Alexa Fluor 647 Phalloidin (1.3 U; Thermo-Fisher Scientific A22287) to label MF, or DAPI from the ProLong Diamond Antifade Mountant (Thermo-Fisher Scientific) to label the nucleus.

Techniques: Derivative Assay, Sequencing, Expressing, Fluorescence, Marker, Concentration Assay, Generated, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

doi: 10.1016/j.isci.2021.103693

Figure Lengend Snippet:

Article Snippet: For experiments in , cells infected with the different ICU3 biosensors were incubated with an anti-caveolin 3 antibody (1:200, Thermo-Fisher Scientific PA1-066) to label the PM, an anti-ryanodine receptor 2 (RyR2) antibody (1:200, Alomone Labs ARR-002) to label the SR, an Alexa Fluor 647 Phalloidin (1.3 U; Thermo-Fisher Scientific A22287) to label MF, or DAPI from the ProLong Diamond Antifade Mountant (Thermo-Fisher Scientific) to label the nucleus.

Techniques: Recombinant, Software

Journal: iScience

Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

doi: 10.1016/j.isci.2021.103693

Figure Lengend Snippet: Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 [RyR2]), MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.

Article Snippet: Rabbit polyclonal anti RyR2 , Alomone , Cat# ARR-002; RRID: AB_2040184.

Techniques: Derivative Assay, Sequencing, Expressing, Fluorescence, Marker, Concentration Assay, Generated, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

doi: 10.1016/j.isci.2021.103693

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti RyR2 , Alomone , Cat# ARR-002; RRID: AB_2040184.

Techniques: Recombinant, Software

Journal: International Journal of Molecular Sciences

Article Title: Transplantation of Neural Precursors Derived from Induced Pluripotent Cells Preserve Perineuronal Nets and Stimulate Neural Plasticity in ALS Rats

doi: 10.3390/ijms21249593

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: Anti-Ryanodine Receptor 2 , Ryanodine Receptor 2/ICC , Rabbit polyclonal , 1:200 , Alomone Labs.

Techniques: Marker